PROJECT SUMMARY Pre-messenger RNA splicing regulates the maturation of a nascent mRNA, an essential process in gene expression. Pre-mRNA splicing is facilitated and regulated by the spliceosome, a large RNA/protein macromolecular machine. The spliceosome is assembled through a complex, multi-step pathway from 5 small nuclear ribonucleoprotein complexes (snRNPS), however the molecular interactions regulating each step is still unknown. The goal of this proposal is to define the roles of an essential splicing protein Dib1 in the regulation of spliceosome assembly. The work in this proposal addresses the proposed model that the presence of a small thioredoxin-like protein Dib1 in the U4/U6-U5 triple snRNP restricts successful incorporation of the pre- messenger RNA into the active site of the spliceosome, therefore acting as a regulator of spliceosome assembly. The proteins of the U4/U6-U5 triple snRNP complex being studied in this project have been directly linked to the human diseases retinitis pigmentosa and Burn-McKeown syndrome. Thus, understanding the function of Dib1 will provide direct insight into the cause of these diseases. Using the model organism, Saccharomyces cerevisiae, the first aim will determine whether Dib1 is a regulatory element of splicing by characterizing the effects of a collection of Dib1 mutants on protein stability, spliceosome assembly, splicing and cell growth. The second aim will determine the temporal relationship and distance between Dib1 and pre- mRNA on the assembling spliceosome using single molecule fluorescence techniques. Successful completion of these studies will identify a novel regulatory element in spliceosome assembly. Moreover, this research will contribute a new understanding of Dib1, which has been linked to other aspects of gene expression including mitosis and transcription, but about which little is known.